The present invention relates generally to apparatus and methods for performing mass spectrometric analyses of material samples and, more particularly, to an improved technique for dissociating parent ions into daughter ions in tandem mass spectrometers.
Mass spectrometry is an analytical technique which relies on the production of ionized fragments from a material sample and subsequent quantification of the fragments based on mass and charge. Typically, positive or negative ions are produced from the sample and accelerated to form an ion beam. Differing mass fractions within the beam are then selected using a mass analyzer, such as a single-focusing or double-focusing magnetic mass analyzer, a time-of-flight mass analyzer, a quadrupole mass analyzer, or the like. A spectrum of fragments having different masses can then be produced, and the identity of compound(s) within the material sample identified based on the spectrum.
An improved form of mass spectrometry, referred to as tandem or MS/MS mass spectrometry has been developed where a mass-selected ion beam (referred to as the parent ion stream) produced by a first mass analyzer is dissociated into a plurality of daughter ion fragments. The daughter ion fragments are then subjected to a second stage mass analyzer, allowing mass quantification of the various daughter ion fractions. Such tandem mass spectrometry has been found to provide much more information on the material being analyzed and to allow for improved discrimination between various species that may be present in a particular sample. Tandem mass spectrometry is discussed in more detail in McLafferty (1981) Science 214:280-287 and Kondrat and Cooks (1978) Anal. Chem. 50:81A-92A.
The present invention is concerned primarily with methods and apparatus for dissociating the parent ion beam into a beam of daughter ions. Collision-induced dissociation (CID) is generally employed to reduce the parent ions into the daughter ions. In the predominant technique, the mass-selected parent ions are collided with gas particles, such as helium or hydrogen particles, to convert a portion of the translational energy into internal excitation energy. A number of the excited molecules will then undergo rapid unimolecular dissociation into structurally significant fragment ions, referred to as daughter ions.
The use of a gas to induce collisional dissociation has several drawbacks. First, very small sample sizes, on the order of micrograms, are generally too small to provide sufficient parent ions to produce a significant stream of daughter ions. Second, the daughter ions are frequently produced over a very large energy range as a result of the kinetics of the gas collision. Such a large energy spread may necessitate the use of double focusing analyzers to obtain sufficient resolution of the daughter ion spectrum. Even with the best performing tandem equipment, however, the highest practical resolution is usually limited to about 1000 daltons because of the signal loss resulting from the broad energy differential. See, e.g., Johnson and Biemann (1987) Biochem. 26:1209-1214. The introduction of a collision gas can also raise the pressure in the mass spectrometer which can result in poor resolution of high mass, e.g., greater than 1000 d, compounds. See, e.g., Aberth (1986) Anal. Chem. 58:1221-1225. Finally, tandem mass spectrometers using electrostatic energy analyzers often display uncertainty in mass calibration as a result of collision-associated translational energy loss. See, e.g., Bricker and Russell (1986) J. Am. Chem. Soc. 108:6174-6179.
To at least partially overcome these problems, a technique referred to as surface-induced dissociation (SID) has been introduced. See, e.g., Mabud et al. (1985) Int. J. Mass Spectrom. Ion Processes 67:285-294; Dekrey (1985) Int. J. Mass Spectrom. Ion Processes 67:295-303; Bier et al. (1977) Int. J. Mass Spectrom. Ion Processes 77:31-47; and Schey et al. (1987) Int. J. Mass Spectrom. Ion Processes 77:49-61. The technique involves colliding a mass-selected low kinetic energy (less than 200 eV) molecular ion beam off a smooth metal surface and mass analyzing the resulting fragments. The object of the technique is to increase the energy transferred to the parent ions to improve their fragmentation efficiency and to avoid certain of the disadvantages of the gas CID method. Unfortunately, SID also suffers from a number of drawbacks. While large amounts of energy can be transferred to the molecular ions for effective fragmentation, the method is only successful with relatively low mass (less than 250 d) hydrocarbons and no fragmentation spectra of biocompounds have yet been reported. In particular, the method has been ineffective with high mass biological polymers, such as proteins, carbohydrates, and polynucleotides, because the high collision energy degrades the individual monomer units, rendering analyses difficult or impossible. Additionally, the collection efficiency (mass of daughter ions collected/mass of parent ions) with tandem mass spectrometers employing SID is relatively low, seldom exceeding 5%. Such low collection efficiency requires use of a larger sample size, which may not always be available. Finally, SID requires about a 90 degree difference between the direction of the incoming parent ion beam and that of the reflected daughter ion beam. As practically all exiting tandem spectrometers use a gas cell for collisional dissociation, they require an in-line geometry between the incoming parent and outgoing daughter beams. Thus, substitution or retrofitting of SID apparatus will require radical restructuring of existing instruments.
For the above reasons, it would be desirable to provide improved methods for collision-induced dissociation of mass-selected ion beams in tandem mass spectrometers. In particular, it would be desirable to maintain the relatively high energy associated with conventional SID methods, while allowing application to a broad range of compounds, including relatively high molecular weight biological compounds. Moreover, it is desired that it be readily adaptable to existing tandem instrument designs where the first and second mass analyzers are aligned in an in-line geometry. Finally, it would be particularly desirable to provide CID with relatively high collection efficiencies, preferably about 10%.